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Secondary antibodies recognize an antibody as their target antigen, usually referred to as the primary antibody which in turn is bound to its antigen or protein of interest. Unlike anti-idiotypic antibodies which bind specifically to the antigen-binding domain, secondary antibodies are specific to the conserved regions of an antibody which are constant within a particular class of antibody such as human IgG or mouse IgM.  Secondary antibodies are therefore able to bind to ranges of antibody clones making them very effective research tools.


Secondary Antibody Selection Tool Protocols and Resources

Here we describe the applications of secondary antibodies and the considerations needed in their selection. 


Applications of Secondary Antibodies

1. Detection of a primary antibody:
 

Detection of a primary antibodySecondary antibodies are commonly used to detect and visualize the presence of a primary antibody in applications like western blotting or immunofluorescent histology. In these applications the secondary antibody is labeled with a reporter molecule, which may be an enzyme like HRP or a fluorophore such as FITC. Multiple secondary antibodies can bind to a single primary antibody increasing the sensitivity and amplifying the signal.

Further signal amplification can be achieved by using an unlabeled secondary and a labeled tertiary antibody if required.

Read our reference guide for essential information on experimental design.

2. Antibody capture:
 

Antibody capture:Unlabeled secondary antibodies can be used to capture antibodies of interest from biological solutions by quantitative ELISA. For example, an unlabeled secondary mouse anti-human IgG may be used as the capture antibody to bind human IgG from a patient serum sample. This is then detected with a labeled secondary mouse anti-human IgG which binds to the captured IgG.

The use of a calibration curve of known standards allows this signal to be quantified.

3. Detection and quantification of recombinant proteins:
 

Secondary antibodies can be used to detect and quantify recombinant proteins that have been engineered to contain antibody domains, for example for ease of expression, detection, stability or increased in vivo half-life.

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Secondary Antibodies at a Glance

The secondary reagents have been carefully selected to provide optimum quality and flexibility for your experimental design:

  • Choose monoclonal or polyclonal
  • IgG molecules or F(ab’)2 fragments
  • Available in many formats
  • Suitable for a wide range of applications such as flow cytometry, western blotting and immunocytochemistry
  • Cross-absorbed antibodies
  • Fluorescent StarBright Secondary Antibodies. Exceptionally bright and ideal for multiplex western blotting

Target

Host

Class and Chain

Conjugates

Human

Mouse

Rat

Goat

Rabbit

Sheep

Bovine

Guinea pig

Cat

Chicken

Monkey

Fish

Dog

Hamster

Horse

Pig

Donkey

Rabbit

Goat

Mouse

Sheep

Rat

Human

Ig

IgA, IgA A1, IgA A2, IgA H, IgA secretory chain

IgD, IgD H, IgD D

IgE

IgG, IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgG H/L, IgG Fc, IgG Fc CH2 domain, IgG CH2 domain, IgG CH3 domain, IgG gamma, IgG Fab, IgG F(ab’)2

IgM, IgM Mu, IgM H

IgG/A/M

Lamba light chain

Kappa light chain

J chain

Purified

Serum

S/N

FITC

Tritc

HRP

Biotin

Alkaline phosphatase

Texas Red

StarBright Blue

DyLight 405

DyLight 488

DyLight 549

DyLight 649

DyLight 680

DyLight 800

Bgal


Secondary Antibody Selection Tool Protocols and Resources

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